WO2008046909A1 - Inhibiteur de l'histone désacétylase, la toxine d'helminthosporium carbonum, pour la suppression de qualité maligne de cellules de neuroblastome - Google Patents

Inhibiteur de l'histone désacétylase, la toxine d'helminthosporium carbonum, pour la suppression de qualité maligne de cellules de neuroblastome Download PDF

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WO2008046909A1
WO2008046909A1 PCT/EP2007/061206 EP2007061206W WO2008046909A1 WO 2008046909 A1 WO2008046909 A1 WO 2008046909A1 EP 2007061206 W EP2007061206 W EP 2007061206W WO 2008046909 A1 WO2008046909 A1 WO 2008046909A1
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groups
group
alkyl
linear
branched
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PCT/EP2007/061206
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Hedwig Deubzer
Olaf Witt
Frank Westermann
Gabriele Becker
Volker Ehemann
Gabi Roenndahl
Ralf Heinrich
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Dkfz Deutsches Krebsforschungszentrum
Ruprecht-Karls-Universität Heidelberg
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
    • C07K5/126—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to treating a subject who has a small round blue cell tumor with a histone deacetylase inhibitor, particularly with an inhibitor that is Helminthosporium carbonum toxin.
  • Helminthosporium carbonum toxin reverses in nanomolar dosage tumor cells from malignant towards benign. Conversion to benign is associated with activation of retinoblastoma tumor suppression networks.
  • Stimulating tumor suppression pathways presents a novel approach to small round blue cell tumor therapy.
  • NB neuroblastoma
  • cytotoxic chemo therapeutic agents target tumor cell growth by (i) causing DNA damage, (ii) interfering with the mitotic spindle organization or (iii) modulating enzymatic activity involved in ribonucleotide synthesis and DNA replication.
  • the underlying principle of all such approaches inadvertently affects not only cancer cells but also untransformed dividing cells, which results in increased toxicity to normal tissue. In the case of NB, it considerably contributes to long-term complications in 44 % of the survivors of high-risk NB . Not bypassing neoplastic mechanisms but activating tumor suppression pathways presents a novel approach to NB therapy.
  • RB tumor suppressor RB
  • E2F transcription factor family RB-Skp2-p27 tapl pathway
  • RB In neuronal tissues, RB not only controls mitotic processes but also governs cell fate specification and differentiation during neurogenesis (37-41). This notion is supported by the ability of RB to bind a broad spectrum of varying proteins including tissue-specific transcription factors (42). RB is known to play some role in neuronal development orchestrating and coupling the complex processes of cell division and maturation (43).
  • RB function is impaired in numerous human cancers of all age classes by different oncogenic events (21).
  • neuroblastoma a clear correlation was identified between poor patient prognosis and deregulated E2F effector cascades (22) as well as an impaired RB-Skp2-p27 tapl pathway (Westermann et al, unpublished data).
  • Neuroblastoma originates from progenitors of mature sympathoadrenal cells and is characterized by marked cellular heterogeneity (2).This is reflected by considerably diverse clinical behaviors with at least three different courses including spontaneous regression of clinically apparent NB, differentiation into mature ganglioneuroma and, finally, aggressive NB characterized by progression despite state-of-the-art multimodal therapy.
  • An overall survival rate of only 34 % in stage IV NB emphasizes the need for novel more biologically based therapeutic strategies activating pathways known to mediate tumor suppression.
  • HDACIs histone deacetylase inhibitors
  • the present invention is a method of treating a subject having a tumor, comprising administering a dose of a histone deacetylase (HDAC) inhibitor to a subject, wherein the HDAC inhibitor activates tumor suppression signalling networks.
  • HDAC histone deacetylase
  • the present invention also encompasses the use of a HDAC inhibitor for the preparation of a medicament for the treatment of tumors, wherein the HDAC inhibitor activates tumor suppression signalling networks.
  • the tumor suppression pathway is a retinoblastoma (RB) tumor suppression pathway.
  • the HDAC inhibitor is selected from a group consisting of carboxylates, hydroxamic acid derivatives, benzamides and cyclic peptides, in particular tetracyclic peptides.
  • the HDAC inhibitor is Helminthosporium Carbonum toxin.
  • the HDAC inhibitor is a compound according to the general formula (I)
  • A is a 9- to 15-membered heterocycle optionally carrying one or more double bonds, and comprising 3 to 5 units of the type -D-E-G- wherein
  • D is selected from -N(R 1 )-, -O-, and -S(O)(R 2 R 3 ) m -; and/or
  • E is selected from methylene -CH 2 -, ethylene -CH 2 CH 2 - and propylene -CH 2 -CH 2 - CH 2 -, wherein in the methylene, ethylene and propylene groups one or more H atoms can be replaced by: linear and branched Ci- to C 10 alkyl groups, which are optionally substituted by one or more substituents from the group -OH, F, -SH, -
  • G is a keto group -C(O)- or a thioketo group -C(S)-; and/or wherein the units -D-E-G- not attached to L can be fully or partially identical with or entirely different from each other; and/or
  • the cycle A can comprise one or more double bonds; and/or
  • L is attached to D or E in one unit -D-E-G- and is a linear C3- to Cs-alkyl group in which one or more carbon atoms may be replaced by non adjacent O atoms, and wherein one or more H atom in the alkyl chain may be substituted by one or more alkyl groups; and/or
  • M is selected from epoxy groups hydroxyamino groups -N(OH)H, hydroxymethyl groups -CH 2 OH, 1-fluoroethyl groups -CHFCH 3 and 1-chloroethyl groups -CHClCH 3 ; and/or
  • R 1 is selected from: H; linear and branched Ci- to C 10 alkyl groups, which are optionally substituted by one or more OH and/or one or more F; and linear or branched C 2 - to C 10 alkylene groups which are optionally substituted by one or more OH and/or one or more F; or R 1 forms together with one alkyl or alkylene group in E a 5- to 7-membered cycle which includes the C and the N atom to which the named groups are attached; and/or
  • R 2 and R 3 are independently of each other selected from: H; linear and branched Ci- to Cio alkyl groups which are optionally substituted by one or more OH and/or one or more F; and linear and branched C 2 - to C 10 alkylene groups, which are optionally substituted by one or more OH and/or one or more F; or R 2 and R 3 , together with the sulfur atom to which they are attached, form a 5- to 7-membered cycle; or one of R 2 , R 3 forms together with one alkyl or alkylene group in E a 5- to 7-membered cycle, which includes the C and the N atom to which the named groups are attached; and/or
  • n 0 or 1
  • the cycle A comprises 3, 4 or 5 amino acids.
  • Suitable amino acids are known to the person skilled in the art and comprise natural amino acids and synthetic amino acids.
  • Natural amino acids are those occurring in nature and comprise: glycine, alanine, serine, cysteine, phenylalanine, tyrosine, tryptophane, threonine, methionine, valine, proline, leucine, iso leucine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine.
  • the named amino acids are found in the L form in nature. In the context of the present invention, the above referenced natural amino acids in the L-form and in the D-form are preferred.
  • the double bonds in the cycle A may be a result of enol forms resulting from tautomerism.
  • the enol forms may be stabilised or fixed by alkylation. Suitable alkylation methods are known to the person skilled in the art.
  • the present invention also includes prodrugs of the compounds of formula (I).
  • suitable prodrugs include molecules according to formula (I) as defined beforehand, and wherein the epoxy function is replaced by an appropriate precursor function therof, e.g. a vicinal diol function, a chlorohydrine function - CH(OH)(Cl)CH- or an olef ⁇ nic double bond.
  • A is a 11- to 13-membered cycle; comprises 4 units of the type -D-E-G- , wherein
  • D is NR 1 ;
  • E is -CH 2 -, wherein one or both H atoms can be replaced by: a linear or branched Ci- to C 4 alkyl group, which are optionally substituted by one or more substituents from the group -OH, F, -SH, -SCH 3 , -NH 2 , -NHC(NH)NH 2 , -C(O)OH, -C(O)NH 2 , C 6 Hs, C 6 H 4 OH, indolyl, imidazolyl or a salt thereof; and/or a linear or branched C 2 - to C 4 alkylene group, which is optionally substituted by one or more OH and/or one or more F; or the named alkyl and alkylene groups form together with the group R 1 a 5- to 7-membered cycle which includes the C and the N atom to which the named groups are attached; and/or G is a keto group -C(O)-; and/or
  • the cycle A can comprise one or more double bonds; and/or
  • R 1 is selected from: H; linear and branched Ci- to C 4 alkyl groups, which are optionally substituted by one or more OH and/or one or more F; and linear and branched C 2 - to C 4 alkylene groups which are optionally substituted by one or more OH and/or one or more F; or R 1 forms together with one alkyl or alkylene group in E a 5- to 7-membered cycle, which includes the C and the N atom to which the named groups are attached, and/or
  • L is attached to E in one unit -D-E-G- and is a linear C3- to Cs-alkyl group in which one or more carbon atoms may be replaced by non adjacent O atoms, and wherein one or more H atom in the alkyl chain may be substituted by one or more alkyl groups; and/or
  • M is selected from epoxy groups and hydroxyamino groups -N(OH)H.
  • the cycle A comprises 4 natural amino acids in the L- or D-form, selected from those cited beforehand.
  • A is a 12-membered cycle and comprises 4 units of the type -D-E-G- , wherein
  • D is NR 1 ;
  • E is -CH 2 -, wherein in one or two units -D-E-G-, one or both H atoms can be replaced by: a linear or branched Ci- to C 4 alkyl group and/or a linear or branched C 2 - to C 4 alkylene group; and wherein in only one unit -D-E-G-, one H atom in - CH 2 - is replaced by a linear or branched Ci- to C 4 alkyl group or a linear or branched C 2 - to C 4 alkylene group, with the said alkyl or alkylene group forming together with R 1 in the neighbouring NR 1 group a 5- to 7-membered cycle, which includes the C and the N atom to which the named groups are attached; and/or
  • G is a keto group -C(O)-;
  • the cycle A can comprise one or more double bonds; and/or
  • R 1 is selected from: H; linear and branched Ci- to C 4 alkyl groups, which are optionally substituted by one or more OH and/or one or more F; and linear and branched C 2 - to C 4 alkylene groups, which are optionally substituted by one or more OH and/or one or more F; and wherein in exactly one unit -D-E-G-, R 1 forms together with the said alkyl or alkylene in the neighbouring -CH 2 - group a 5- to 7- membered cycle, which includes the C and the N atom to which the named groups are attached; and/or
  • L is attached to E in one unit -D-E-G- and is a linear C3- to Cs-alkyl group, and wherein one or more H atom in the alkyl chain may be substituted by one or more alkyl groups; and/or
  • M is selected from epoxy groups and hydroxyamino groups -N(OH)H; and/or
  • the cycle A comprises 4 natural amino acids in the L- or D-form.
  • Embodiments, which are still preferred over those under C, are classified below under D:
  • A is a 12-membered cycle and comprises 4 units of the type -D-E-G- , wherein D is NR 1 ; and/or
  • E is -CH 2 -, wherein in one or two units -D-E-G-, one or both H atoms can be replaced by: a methyl or ethyl group; and wherein in only one unit -D-E-G-, one H atom in -CH 2 - is replaced by a linear or branched Ci- to C 4 alkyl group or a linear or branched C 2 - to C 4 alkylene group, and wherein the said alkyl or alkylene group forms together with R 1 in the neighbouring NR 1 group a 5- to 7-membered cycle which includes the C and the N atom to which the named groups are attached;
  • G is a keto group -C(O)-;
  • the cycle A can comprise one or more double bonds; and/or
  • R 1 is H or a linear or branched Ci- to C4-alkyl group in three of the units -D-E-G-; and wherein in the other unit -D-E-G, R 1 forms together with the said alkyl or alkylene in the neighbouring -CH 2 - group a 5- to 7-membered cycle, which includes the C and the N atom to which the named groups are attached; and/or
  • L is attached to E in one unit -D-E-G- and is a C3- to Cs-alkyl group, and wherein one or more H atom in the alkyl chain may be substituted by one or more alkyl groups; and/or
  • M is selected from epoxy groups and hydroxyamino groups -N(OH)H; and/or
  • the cycle A comprises 4 natural amino acids in the L- or D-form.
  • the compound of the general formula (II) is known as such and does as a compound not belong to the present invention, only with respect to its particular pharmacological and biological properties, as laid out hereinafter.
  • the present invention relates to a compound according to formula (I) as defined above in general form or in preferred embodiments, or a pharmaceutically acceptable salt thereof, for use as medicament.
  • the present invention relates to a compound according to formula (I) as defined above in general form or in preferred embodiments, or a pharmaceutically acceptable salt thereof, for the manufacture of a pharmaceutical composition for the treatment of the tumors or cancer diseases recited herein and, preferably, the Small Round Blue Cell Tumor.
  • Small Round Blue Cell Tumor is, preferably, a pediatric soft tissue tumor.
  • a Small Round Cell Tumor as usd herein is a tumor selected from the group consisting of: Desmoplastic small round blue cell tumor, Ewing's Sarcoma, Acute Leukemia, Small Cell Lung Carcinoma, Small Cell Mesothelioma, Neuroblastoma, Primitive Neuroectodermal tumor, Rhabdomyosarcoma, WiIm' s Tumor, and Melanoma, most preferably, it is a neuroblastoma.
  • the tumor suppression pathway is the retinoblastoma tumor suppression pathway.
  • the cancer is a Small Round Blue Cell Tumor.
  • the Small Round Blue Cell Tumor is selected from the group consisting of Desmoplastic small round blue cell tumor, Ewing's Sarcoma, Acute Leukemia, Small Cell Lung Carcinoma, Small Cell Mesothelioma, Neuroblastoma, Primitive Neuroectodermal tumor, Rhabdomyosarcoma, Wilm's Tumor, and Melanoma.
  • the Small Round Blue Cell Tumor is a Neuroblastoma.
  • the subject is a mammal.
  • the mammal is a human, primate, rat, dog, cat, cattle, mouse, guinea pig, gerbil, pig, or sheep.
  • the subject is a human.
  • the dose of the HDAC inhibitor administered to the subject is about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM, about 26 nM, about 27 nM, about 28 nM, about 29 nM, about 30 nM, or more than about 30 nM, or any integer there in between.
  • the HDAC inhibitor which is administered at this dose is a peptide according to formula (I), preferably a tetrapeptide, in particular Helminthosporium Carbonum toxin.
  • the dose of the HDAC inhibitor administered to the subject is between 1-5 nM, between 5-10 nM, between 10-15 nM, between 15-20 nM, between 20-25 nM, between 25-30 nM or more than 30 nM, e.g., between 30-40 nM or 40-50 nM.
  • the dose is between 10-20 nM.
  • the HDAC inhibitor which is administered to the subject is Helminthosporium Carbonum toxin at a dose concentration of between 10-2O nM.
  • Figures IA- ID HC-toxin efficiently mediates growth arrest and apoptosis in NB cells with MYCN single copy status and with transfected human MYCN oncogene governing enhanced expression.
  • Figure IA Time-course and dose-response growth curves of SH-EP NB cells cultured with HC-toxin or MeOH solvent control. Induction of histone 4 acetylation (panAc-H4) by HC-toxin is shown in the upper panel.
  • Figure IB induction of G 0 ZG 1 cell cycle arrest and apoptosis (sub- Gi) in SH-EP NB cells by HC-toxin.
  • FIG. 1C Shown at right are exemplarily FACS profiles of solvent control and HC-toxin treated cells after 72 h of exposure. Striped areas represent S phase cells.
  • Figure 1C Tet21N, a synthetic N-Myc protein inducible expression system on the basis of the tetracycline repressor of Escherichia coli, with ⁇ -actin serving as loading control.
  • Figure ID WAC-2 and SH-S Y5 Y NB cells.
  • Figures 2 A and 2B HC-toxin sustainably mediates growth arrest and apoptosis in NB cells with MYCN oncogene amplification.
  • Figure 2A Time course dose- response growth curves of BE(2)-C NB cells cultured with HC-toxin or MeOH solvent control.
  • Figure 2B Induction of GoZGi cell cycle arrest in the diploid and tetraploid cell fraction of BE(2)-C NB cells by HC-toxin.
  • Figures 3A and 3B HC-toxin significantly evokes growth arrest and apoptosis in primary neuroblasts.
  • Figure 3A Induction of primary neuroblast cell death by HC- toxin ⁇ lower panel) in a short term culture of stage IV MYCN single copy neuroblasts derived from bone marrow ⁇ upper panel) of a 2 year and 10 months old child compared to MeOH solvent treated controls ⁇ middle panel).
  • Figure 3B GoZGi cell cycle arrest and apoptosis mediated by HC-toxin ⁇ lower panels) in primary MYCN single copy neuroblasts ⁇ left upper pane! derived from the stage I NB of a seven months old infant ⁇ right upper panel).
  • Figures 4A-4D HC-toxin potently induces neuronal differentiation and loss of neuroectodermal stem cell marker expression in NB cells.
  • Figure 4A Neurite outgrowth in SH-EP and BE(2)-C NB cells following treatment with 15 and 20 nM Helminthosporium carbonum (HC)-toxin or MeOH solvent control for 72 h or 14 d, respectively.
  • Figure 4B Up-regulation of neuronal marker genes on mRNA level.
  • Quantitative ,,real time" RT-PCR analysis revealed an induction of neurofilament 3 ⁇ NEF3, 150 kDa medium), tyrosine hydroxylase (TH), microtubule associated protein 2 ⁇ MAPI), synaptophysin 1 ⁇ SYP1) and synapsin 1 ⁇ SYN1) in HC-toxin treated SH-EP and an induction of the dendritic and synaptic markers in BE(2)-C NB cells compared to respective controls.
  • Figure 4C Down- regulation of the intermediate filament nestin on mRNA level.
  • Figure 4D Up- regulation of neuronal markers on protein level.
  • Immunocytochemistry showed an increased expression or induction of neurofilament 3 (anti-Nef 3, 150 kDa medium), tyrosine hydroxylase (anti-TH), microtubule associated protein 2 (anti- MAPI) after 12O h and of synaptophysin 1 (anti-SYPl) and synapsin 1 (anti- SYNl) after 200 h of treatment in SH-EP NB cells, respectively.
  • FIGs 5A and 5B HC-toxin strongly reduces anchorage-independent growth and invasive potential of NB cells.
  • Figure 5 A Influence of HC-toxin upon anchorage- independent transformation potential. Pretreated WAC-2 and BE(2)-C NB cells were plated in soft agar (see Example 1).
  • Figure 5B BE(2)-C (Al-4) and WAC-2 (Bl-4) NB cell invasiveness and extracellular matrix (ECM) degradation was assessed using Boyden chambers. Prior to seeding cells into the upper compartment of the chambers, cells were treated for 5 days with 20 nM HC-toxin or MeOH solvent control. To adjust for differences in proliferation and migratory potential, normalization was obtained using Boyden chambers with control inserts (Al, A2, Bl, B 2) (see Example 1).
  • FIGS 6 A and 6B HC-toxin sustainably activates RB tumor suppression pathways in NB cells.
  • Figure 6A Activation of RB tumor suppressor function by HC-toxin (HCT) resulting in profound reduction of E2F1 and E2F target genes (Survivin, Mad2, N-myc) as well as in activation of the Skp2-p27 tapl pathway in BE(2)-C NB cells. Shown also is the induction of p21 cipl/waf l by HC-toxin.
  • Figure 6B HC-toxin mediated induction of pl5 INK4b and pl6 INK4a .
  • Figures 7 A and 7B HC-toxin promotes no detectable effects upon viability, RB mediated cell cycle control and apoptosis of primary human skin fibroblasts (PHSFs).
  • Figure 7A Time course dose-response growth curves of PHSFs cultured with HC-toxin or methanol (MeOH) solvent control.
  • Figure 7B No detectable effect of HC-toxin upon cell cycle, apoptosis (sub-Gi) and granularity (middle panel) of PHSFs.
  • Figure 7C No detectable HC-toxin mediated modulation of RB tumor suppressor activity.
  • HDAC inhibitors which are compounds according to the general formula (I)
  • the compounds can be tri-, tetra or pentapeptides, preferably tetrapeptides.
  • the symbols have the following meanings: A:
  • A is a 9- to 15-membered heterocycle optionally carrying one or more double bonds, and comprising 3 to 5 units of the type -D-E-G- wherein
  • D is selected from -N(R 1 )-, -O-, and -S(O)(R 2 R 3 ) m -; and/or
  • E is selected from methylene -CH 2 -, ethylene -CH 2 CH 2 - and propylene -CH 2 -CH 2 - CH 2 -, wherein in the methylene, ethylene and propylene groups one or more H atoms can be replaced by: linear and branched Ci- to C 10 alkyl groups, which are optionally substituted by one or more substituents from the group -OH, F, -SH, - SCH 3 , -NH 2 , -NHC(NH)NH 2 , -C(O)OH, -C(O)NH 2 , C 6 H 5 , C 6 H 4 OH, indolyl, imidazolyl or a salt thereof; and linear and branched C 2 - to C 10 alkylene groups, which are optionally substituted by one or more OH and/or one or more F; or, in case 2 or more even-numbered substituents are present, the named alkyl or alkylene group forms together with a further al
  • G is a keto group -C(O)- or a thioketo group -C(S)-;
  • the cycle A can comprise one or more double bonds; and/or L is attached to D or E in one unit -D-E- G- and is a linear C 3 - to Cs-alkyl group in which one or more carbon atoms may be replaced by non adjacent O atoms, and wherein one or more H atom in the alkyl chain may be substituted by one or more alkyl groups; and/or
  • M is selected from epoxy groups hydroxyamino groups -N(OH)H, hydroxymethyl groups -CH 2 OH, 1-fluoroethyl groups -CHFCH 3 and 1-chloroethyl groups -CHCICH 3 ; and/or
  • R 1 is selected from: H; linear and branched Ci- to C 10 alkyl groups, which are optionally substituted by one or more OH and/or one or more F; and linear or branched C 2 - to C 10 alkylene groups which are optionally substituted by one or more OH and/or one or more F; or R 1 forms together with one alkyl or alkylene group in E a 5- to 7-membered cycle which includes the C and the N atom to which the named groups are attached; and/or
  • R 2 and R 3 are independently of each other selected from: H; linear and branched Ci- to Cio alkyl groups which are optionally substituted by one or more OH and/or one or more F; and linear and branched C 2 - to C 10 alkylene groups, which are optionally substituted by one or more OH and/or one or more F; or R 2 and R 3 , together with the sulfur atom to which they are attached, form a 5- to 7-membered cycle; or one of R 2 , R 3 forms together with one alkyl or alkylene group in E a 5- to 7-membered cycle, which includes the C and the N atom to which the named groups are attached; and/or
  • n 0 or 1
  • L is always attached to E in one of the units -D-E-G-, which unit has to be different from the other units -D-E-G-.
  • the cycle A comprises 3, 4 or 5 amino acids.
  • Suitable amino acids are known to the person skilled in the art and comprise natural amino acids and synthetic amino acids.
  • Natural amino acids are those occurring in nature and comprise: glycine, alanine, serine, cysteine, phenylalanine, tyrosine, tryptophane, threonine, methionine, valine, proline, leucine, isoleucine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine.
  • the named amino acids are found in the L form in nature. In the context of the present invention, the above referenced natural amino acids in the L-form and in the D-form are preferred.
  • the double bonds in the cycle A may be a result of enol forms resulting from tautomerism.
  • the enol forms may be stabilised or fixed by alkylation. Suitable alkylation methods are known to the person skilled in the art.
  • the present invention also includes prodrugs of the compounds of formula (I).
  • suitable prodrugs include molecules according to formula (I) as defined beforehand, and wherein the epoxy function is replaced by an appropriate precursor function therof, e.g. a vicinal diol function, a chlorohydrine function - CH(OH)(Cl)CH- or an olefinic double bond.
  • A is a 11- to 13-membered cycle; comprises 4 units of the type -D-E-G- , wherein
  • D is NR 1 ;
  • E is -CH 2 -, wherein one or both H atoms can be replaced by: a linear or branched Ci- to C 4 alkyl group, which are optionally substituted by one or more substituents from the group -OH, F, -SH, -SCH 3 , -NH 2 , -NHC(NH)NH 2 , -C(O)OH, -C(O)NH 2 , C 6 Hs, C 6 H 4 OH, indolyl, imidazolyl or a salt thereof; and/or a linear or branched C 2 - to C 4 alkylene group, which is optionally substituted by one or more OH and/or one or more F; or the named alkyl and alkylene groups form together with the group R 1 a 5- to 7-membered cycle which includes the C and the N atom to which the named groups are attached; and/or
  • G is a keto group -C(O)-;
  • the units -D-E-G- not attached to L can be fully or partially identical with or entirely different from each other; and/or the cycle A can comprise one or more double bonds; and/or
  • R 1 is selected from: H; linear and branched Ci- to C 4 alkyl groups, which are optionally substituted by one or more OH and/or one or more F; and linear and branched C 2 - to C 4 alkylene groups which are optionally substituted by one or more OH and/or one or more F; or R 1 forms together with one alkyl or alkylene group in E a 5- to 7-membered cycle, which includes the C and the N atom to which the named groups are attached, and/or
  • L is attached to E in one unit -D-E-G- and is a linear C3- to Cs-alkyl group in which one or more carbon atoms may be replaced by non adjacent O atoms, and wherein one or more H atom in the alkyl chain may be substituted by one or more alkyl groups; and/or
  • M is selected from epoxy groups and hydroxyamino groups -N(OH)H.
  • the cycle A comprises 4 natural amino acids in the L- or D-form, selected from those cited beforehand.
  • A is a 12-membered cycle and comprises 4 units of the type -D-E-G- , wherein
  • D is NR 1 ;
  • E is -CH 2 -, wherein in one or two units -D-E-G-, one or both H atoms can be replaced by: a linear or branched Ci- to C 4 alkyl group and/or a linear or branched C 2 - to C 4 alkylene group; and wherein in only one unit -D-E-G-, one H atom in - CH 2 - is replaced by a linear or branched Ci- to C 4 alkyl group or a linear or branched C 2 - to C 4 alkylene group, with the said alkyl or alkylene group forming together with R 1 in the neighbouring NR 1 group a 5- to 7-membered cycle, which includes the C and the N atom to which the named groups are attached; and/or G is a keto group -C(O)-; and/or
  • the cycle A can comprise one or more double bonds; and/or
  • R 1 is selected from: H; linear and branched Ci- to C 4 alkyl groups, which are optionally substituted by one or more OH and/or one or more F; and linear and branched C 2 - to C 4 alkylene groups, which are optionally substituted by one or more OH and/or one or more F; and wherein in exactly one unit -D-E-G-, R 1 forms together with the said alkyl or alkylene in the neighbouring -CH 2 - group a 5- to 7- membered cycle, which includes the C and the N atom to which the named groups are attached; and/or
  • L is attached to E in one unit -D-E-G- and is a linear C3- to Cs-alkyl group, and wherein one or more H atom in the alkyl chain may be substituted by one or more alkyl groups; and/or
  • M is selected from epoxy groups and hydroxyamino groups -N(OH)H; and/or
  • the cycle A comprises 4 natural amino acids in the L- or D-form.
  • Embodiments, which are still preferred over those under C, are classified below under D:
  • A is a 12-membered cycle and comprises 4 units of the type -D-E-G- , wherein
  • D is NR 1 ;
  • E is -CH 2 -, wherein in one or two units -D-E-G-, one or both H atoms can be replaced by: a methyl or ethyl group; and wherein in only one unit -D-E-G-, one H atom in -CH 2 - is replaced by a linear or branched Ci- to C 4 alkyl group or a linear or branched C 2 - to C 4 alkylene group, and wherein the said alkyl or alkylene group forms together with R 1 in the neighbouring NR 1 group a 5- to 7-membered cycle which includes the C and the N atom to which the named groups are attached;
  • G is a keto group -C(O)-;
  • the cycle A can comprise one or more double bonds; and/or
  • R 1 is H or a linear or branched Ci- to C4-alkyl group in three of the units -D-E-G-; and wherein in the other unit -D-E-G, R 1 forms together with the said alkyl or alkylene in the neighbouring -CH 2 - group a 5- to 7-membered cycle, which includes the C and the N atom to which the named groups are attached; and/or
  • L is attached to E in one unit -D-E-G- and is a C3- to Cs-alkyl group, and wherein one or more H atom in the alkyl chain may be substituted by one or more alkyl groups; and/or
  • M is selected from epoxy groups and hydroxyamino groups -N(OH)H; and/or
  • the cycle A comprises 4 natural amino acids in the L- or D-form.
  • formula (II) i.e. Helminthosporium carbonum toxin (HC-Toxin).
  • the present invention demonstrates that Helminthosporium carbonum (HC)-toxin, an inhibitor of histone deacetylases (HDACs), induces G 0 ZG 1 cell cycle arrest, blockage of DNA-replication and apoptosis in neuroblastoma cell lines with and without MYCN oncogene amplification, as well as in primary neuroblasts. Only nanomolar concentrations of HC-toxin are required to achieve this result. Cell cycle exit is followed by neuronal differentiation and loss of neuroectodermal stem cell marker expression as well as by decrease in anchorage-independent plating efficiency and invasiveness in vitro.
  • HDACs histone deacetylases
  • NB Neuroblastoma
  • RB retinoblastoma
  • p2l cipl/waf ⁇ V RB, pl5 -I 1 N N K K 4 4 b B Z / RB and pl6 -I 1 N N K K 4 4 a a Z RB mediated cell cycle control is enforced by HC- toxin.
  • conversion of NB cells from malignant towards more benign by nanomolar doses of the HDAC inhibitor Helminthosporium carbonum toxin is associated with activation of RB tumor suppression pathways.
  • HDACI HDAC inhibitor
  • HC-toxin Helminthosporium carbonum
  • RB controlled tumor suppression pathways are known to be disabled in numerous human cancers (21).
  • Deregulation of RB signalling pathways such as the RB-E2F and the RB-Skp2-p27 tapl pathways causes impairment of cell cycle exit and thus leads to uncontrolled cell cycle transition (21, 55).
  • RB-E2F and the RB-Skp2-p27 tapl pathways causes impairment of cell cycle exit and thus leads to uncontrolled cell cycle transition (21, 55).
  • E2F target Mad2 22).
  • Aberrant MAD2 transcript levels were found to be linked to high E2F1 messenger RNA levels in a near-linear correlation suggesting RB pathway alterations in NB with poor prognosis (22).
  • Westermann and colleagues identified a clear link between progressing NB and an impaired Skp2-p27 kipl pathway (unpublished data). In benign NB, this pathway was found to be functional.
  • the present inventors hypothesized that conversion of NB cells from malignant towards more benign by nanomolar doses of the HDACI HC-toxin is associated with activation of RB tumor suppression pathways.
  • Western analysis was applied to assess the influence of HC-toxin upon RB tumor suppression signalling pathways in four MYCN amplified NB cell lines. These experiments showed that conversion of NB cells from malignant towards more benign by HC- toxin is associated with activation of RB tumor suppression pathways.
  • HC-toxin was identified in screening experiments to elicit efficiently and with high potency cell cycle arrest and apoptosis in five NB cell lines harboring different MYCN oncogene expression.
  • the present inventors also surmised that suppression of malignant qualities of NB cells by HC-toxin is associated with activation of RB tumor suppression pathways.
  • Inhibition of HDACs by 10 to 20 nM doses of HC-toxin was found to enhance the growth inhibitory function of RB and its control of E2F dependent effectors as well as the Skp2-p27 tapl pathway in four MFCTV-amplified NB cells.
  • p21 cipl/waf" V RB, pl5 INK4b Z RB and pl6 INK4a Z RB mediated cell cycle control was enforced by HC-toxin.
  • reversion of NB cells from malignant towards more benign by HC-toxin is associated with activation of RB tumor suppression pathways.
  • HC-toxin accordingly imposed no unfavorable effects upon these untransformed dividing cells.
  • in vivo animal testing is imperative in respect to the further evaluation of HC-toxin mediated toxicity upon normal tissues as well as in respect to its bioavailability.
  • the present invention uses terms and phrases that are well known to those in the art of molecular biology. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein, and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, microbial culture, cell culture, tissue culture, transformation, transfection, transduction, analytical chemistry, organic synthetic chemistry, chemical syntheses, chemical analysis, and pharmaceutical formulation and delivery.
  • the present invention includes all stereoisomeric forms of the compounds of the formula (I). Centers of asymmetry that are present in the compounds of formula (I) all independently of one another have S configuration or R configuration.
  • the invention includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all ratios.
  • compounds according to the present invention which can exist as enantiomers can be present in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios.
  • the invention includes both the cis form and the trans form as well as mixtures of these forms in all ratios. All these forms are an object of the present invention.
  • the preparation of individual stereoisomers can be carried out, if desired, by separation of a mixture by customary methods, for example by chromatography or crystallization, by the use of stereochemically uniform starting materials for the synthesis or by stereoselective synthesis.
  • a derivatization can be carried out before a separation of stereoisomers.
  • the separation of a mixture of stereoisomers can be carried out at the stage of the compounds of the formula (I) or at the stage of an intermediate during the synthesis.
  • the present invention also includes all tautomeric forms of the compounds of formula (I).
  • the invention also comprises their corresponding pharmaceutically or toxico logically acceptable salts, in particular their pharmaceutically utilizable salts.
  • the compounds of the formula (I), which contain acidic groups can be present on these groups and can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids.
  • Compounds of the formula (I), which contain one or more basic groups i.e.
  • acids which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids.
  • suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art.
  • the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions).
  • the respective salts according to the formula (I) can be obtained by customary methods, which are known to the person skilled in the art like, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts.
  • the present invention also includes all salts of the compounds of the formula (I), which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.
  • the present invention furthermore includes all solvates of compounds of the formula (I), for example hydrates or adducts with alcohols, active metabolites of the compounds of the formula (II), and also derivatives and prodrugs of the compounds of the formula (I) which contain physiologically tolerable and cleavable groups, for example esters, amides and compounds in which the N-H group depicted in formula (I) is replaced with an N-alkyl group, such as N-methyl, or with an N-acyl group, such as N-acetyl or N-argininyl, including pharmaceutically acceptable salts formed on functional groups present in the N- acyl group.
  • physiologically tolerable and cleavable groups for example esters, amides and compounds in which the N-H group depicted in formula (I) is replaced with an N-alkyl group, such as N-methyl, or with an N-acyl group, such as N-acetyl or N-argininyl, including pharmaceutically acceptable salt
  • the compounds of the formula (I) can be purified by customary purification procedures, for example by recrystallization or chromatography.
  • the starting compounds for the preparation of the compounds of the formula (I) are commercially available or can be prepared according to or analogously to literature procedures.
  • the compounds according to the formula (I) can also be used in combination with other pharmaceutically active compounds, preferably compounds which are able to enhance the effect of the compounds according to the general formula (I).
  • Examples of such compounds include: (i) antimetabolites, cytarabine, fludarabine, 5-fluoro-2'-deoxyuridine, gemcitabine, hydroxyurea or methotrexate; (ii) DNA- fragmenting agents, bleomycin, (iii) DNA-crosslinking and alkylating agents, chlorambucil, cisplatin, carboplatin, fotemustine, cyclophosphamide, ifosfamide, dacarbazine or nitrogen mustard; (iv) intercalating agents, adriamycin (doxorubicin) or mitoxantrone; (v) protein synthesis inhibitors, L-asparaginase, cycloheximide, puromycin or diphteria toxin; (vi) topoisomerase I poisons, camptothecin or topotecan; (vii) topoisomerase II poisons, etoposide (VP- 16) or teniposide; (
  • the compounds of the formula (I) and their pharmaceutically acceptable salts can be administered to animals, preferably to mammals, and in particular to humans, as pharmaceuticals by themselves, in mixtures with one another or in the form of pharmaceutical preparations. Further subjects of the present invention therefore also are the compounds of the formula (I) and their pharmaceutically acceptable salts for use as pharmaceuticals, their use as inhibitors of DNMTs and/or DNA methylation, and in particular their use in the therapy and prophylaxis of the above- mentioned syndromes as well as their use for preparing pharmaceuticals for these purposes.
  • subjects of the present invention are pharmaceutical preparations (or pharmaceutical compositions), which comprise an effective dose of at least one compound of the formula (I) and/or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, i.e. one or more pharmaceutically acceptable carrier substances and/or additives.
  • a pharmaceutically acceptable carrier i.e. one or more pharmaceutically acceptable carrier substances and/or additives.
  • the pharmaceuticals according to the invention can be administered orally, for example in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example in the form of suppositories. Administration can also be carried out parenterally, for example subcutaneously, intramuscularly or intravenously in the form of solutions for injection or infusion.
  • Suitable administration forms are, for example, percutaneous or topical administration, for example in the form of ointments, tinctures, sprays or transdermal therapeutic systems, or the inhalative administration in the form of nasal sprays or aerosol mixtures, or, for example, microcapsules, implants or rods.
  • the preferred administration form depends, for example, on the disease to be treated and on its severity.
  • the preparation of the pharmaceutical preparations can be carried out in a manner known per se.
  • one or more compounds of the formula (I) and/or their pharmaceutically acceptable salts together with one or more solid or liquid pharmaceutical carrier substances and/or additives (or auxiliary substances) and, if desired, in combination with other pharmaceutically active compounds having therapeutic or prophylactic action, are brought into a suitable administration form or dosage form, which can then be used as a pharmaceutical in human or veterinary medicine.
  • Carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc.
  • Suitable carriers for the preparation of solutions, for example of solutions for injection, or of emulsions or syrups are, for example, water, physiologically sodium chloride solution, alcohols such as ethanol, glycerol, polyols, sucrose, invert sugar, glucose, mannitol, vegetable oils, etc.
  • Suitable carriers for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid.
  • the pharmaceutical preparations can also contain additives, for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
  • additives for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
  • the dosage of the compound of the formula (I) to be administered and/or of a pharmaceutically acceptable salt thereof depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, it depends on the nature and the severity of the disorder to be treated, and also on the sex, age, weight and individual responsiveness of the human or animal to be treated, on the efficacy and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to compounds of the formula (I).
  • the daily dose can be administered in a single dose or, in particular when larger amounts are administered, be divided into several, for example two, three or four individual doses. In some cases, depending on the individual response, it may be necessary to deviate upwards or downwards from the given daily dose.
  • Human NB cell lines with (BE(2)-C, Kelly, LA-N-I and NGP) and without (SH- EP, SH-SY5Y) MYCN oncogene amplification were cultured according to the depositor's instructions.
  • Tet21N NB cells engineered to variably express tetracycline-controlled N-myc protein and WAC-2 NB cells containing a transfected functional human MYCN oncogene governing enhanced expression were cultured as previously described (23, 24).
  • PHSFs derived from an adult undergoing surgical treatment were grown in DMEM supplemented with 10 % (v/v) fetal bovine serum and 1 % (v/v) 1-glutamine.
  • NB tumor and bone marrow specimens were obtained from patients as part of their routine care following informed consent.
  • Primary neuroblasts (MYCN single copy) isolated from a stage 1 NB of a seven months old infant were grown in DMEM augmented with 1% autologous serum.
  • Stage IV neuroblasts (MYCN single copy) derived from bone marrow metastasis of a 2 year and 10 months old child were isolated by Ficoll-paque (Amersham Bio sciences, Buckinghamshire, UK).
  • CCAACTATGCCATGCTTTGAAG-3' Thermal cycling was conducted under standard conditions. Each sample was analyzed at least in duplicate with high reproducibility. Specificity of resulting amplicons was confirmed by melting curves and agarose gel electrophoresis. Ct- values of target genes were normalized with corresponding C t - value means of the reference genes ACTB, GAPD, ASH2L and HPGD, which remained unaltered under all conditions investigated.
  • Immunofluorescence was performed on cells grown on poly-D-lysine coated glass coverslips placed in 24-well chamber slides (Nunc, Roskilde, Denmark), fixed with 2 % paraformaldehyde (Merck) in 0.1 M PO 4 buffer and subsequently permeabilized with phosphate buffered saline containing 0.1 % Triton X-IOO (Serva) (PBST). Unspecific labeling was blocked with a solution of 10 % normal goat serum, 0.25 % bovine serum albumin dissolved in PBST.
  • Flow cytometric analysis was performed using a Galaxy Pro Flow Cytometer (Partec, Muenster, Germany). Natively sampled cells were prepared in 2.1% citric acid/ 0.5% tween 20 (26) and stained with 50 ⁇ g/ml DAPI dissolved in phosphate buffer (7.2 g Na 2 HPO 4 x 2H 2 O in 100ml distilled water, pH 8.0). DNA-index and cell cycle measurements are based upon the analysis of 30.000 - 100.000 cells. For histogram analyses acquired in the FCS-mode, the Multicycle program (Phoenix Flow Systems, San Diego, CA) was applied. Human lymphocytes from healthy donors served as internal standard for calibrating the diploid DNA-index. The mean coefficient of variations (CV) of these measurements was 0.9.
  • NB cells Growth arrest and apoptosis in NB cells with MYC/V-single copy status and with transfected human MYCN oncogene governing enhanced expression.
  • the present inventors systematically examined a panel of small-molecule HDACIs covering the chemical classes of carboxylates, hydroxamic acid derivatives, benzamides and tetracyclic peptides for their efficacy and potency to elicit anti-tumoral effects in experimental NB.
  • the NB cell lines SH-EP, SH- SY5Y, WAC-2 and BE(2)-C as well as Tet21N NB cells engineered to variably express tetracycline-controlled N-myc protein were used in these screening experiments (data not shown).
  • the cyclic tetrapeptide HC-toxin isolated from the fungus Helminthosporium carbonum was identified to elicit the strongest effects regarding growth arrest and apoptosis as well as neuronal differentiation and loss of neuroectodermal stem cell marker expression at the lowest concentration in 10 to 20 nanomolar range.
  • the present inventors analyzed the effect of HC-toxin upon NB cell proliferation after proving a significant increase in histone 4 acetylation in SH-EP NB cells after 6 h of 10 - 20 nM HC-toxin exposure (Fig. IA, upper panel).
  • Cells were treated with various concentrations of HC-toxin (HCT) and MeOH solvent control (% v/v) for 6 h or remained untreated (ut). Proteins were extracted and analyzed for panAc- H4 by Western analysis with Coomassie stain of the gel serving as loading control.
  • HCT HC-toxin
  • MeOH solvent control % v/v
  • Fig. IB Cell cycle analysis revealed a significant G 0 ZG 1 arrest from 24 h to 72 h of HC-toxin treatment (Fig. IB). Moreover, a strong induction of apoptosis was observed at 48 h and 72 h of HC-toxin treatment (Fig. IB). With respect to Fig. IB, changes in phases of the cell cycle and in apoptosis were analyzed as described in Example 1. After 72 h HC-toxin treatment there was an increase in SH-EP NB cell granularity (Fig. IB, middle panel). In parallel, a profound increase in SH-EP cell granularity was observed (Fig. IB).
  • SH-SY5Y cells subcloned from the same NB tumor as SH-EP (29) but characterized by a neuronal phenotype, are also sensitive to inhibition of proliferation by nanomolar HC-toxin treatment (Fig. ID, right panel).
  • HC-toxin mediates significant inhibition of proliferation at nanomolar concentration in a panel of NB cell lines independent of their phenotype and with particular efficacy against NB cells harboring enhanced MYCN oncogene expression.
  • the physiologically MYCN amplified NB cell line BE(2)-C consisting of a diploid and a tetraploid cell fraction was chosen for analysis.
  • HC-toxin evoked in 10 - 20 nM dosage an increase in histone 4 acetylation after 6 h (Fig. 2A, upper panel).
  • harvested cells were evaluated for number and viability by trypan blue exclusion. Results presented are means ⁇ SDs of three independent experiments performed in triplicates. Induction of histone 4 acetylation (panAc-H4) by HC-toxin is given in the upper panel.
  • stage IV neuroblasts metastasized to bone marrow (Fig. 3 A, upper panel) were apoptotic after 48 h of treatment (Fig. 3 A, lower panel) compared to controls (Fig. 3A, middle panel).
  • HC-toxin treatment of stage I neuroblasts (Fig. 3B, left upper panel) derived from the NB tumor of a 7 months old infant (Fig. 3B, right upper panel) resulted in a significant G 0 ZG 1 arrest and the induction of apoptosis (Fig. 3B, lower panels).
  • Fig. 3B differences compared to MeOH solvent treated controls reached statistical significance (right lower panel) with P ⁇ .05 (*).
  • HC-toxin has similar effects in established NB cell lines and in primary NB cultures.
  • Neuronal differentiation and loss of neuroectodermal stem cell marker expression are characterized by neuronal differentiation and loss of neuroectodermal stem cell marker expression.
  • HC-toxin treated NB cells revealed dose-dependent Iy - apart from structural alterations specific for apoptotic cells - extensive changes in morphology towards a neuronal phenotype. This was characterized by rounded cell bodies, intermediate to long-length fine neurites and extensive interconnections among neurites (Fig. 4A). With respect to Fig. 4A, representative sections were photographed under an inverted phase-contrast microscope (Olympus, 200 x; scale bar: 15 ⁇ m). Some NB cell lines including BE(2)-C responded to HC-toxin treatment with ganglionic- like formations (Fig AA, right lower panel).
  • HC-toxin treated SH-EP cells Approximately 90 - 95 % of HC-toxin treated SH-EP cells (Fig AA, right upper panel) exhibited neuronal differentiation after 72 h of treatment whereas first effects in BE(2)-C cells were observed after 96 h of exposure reaching their maximum after 14 d with interconnections among neurites and pseudoganglionic formations.
  • Quantitative real time RT-PCR analysis revealed a up-regulation of the axonal marker neurofilament 3, 150 kDa
  • the intermediate filament nestin is expressed in multipotent neuroectodermal precursor cells and is down-regulated as these cells divide and differentiate along their respective neural or glial lineages (30).
  • infiltrating cells are characterized by elevated nestin levels (31, 32).
  • nestin is one mediator of N-myc associated aggressiveness in neuroblastoma (33).
  • HC-toxin upon nestin mRNA levels in BE(2)-C NB cells belonging to the most malignant NB cell type (34).
  • Nestin mRNA expression was significantly down- regulated in HC-toxin treated BE(2)-C cells (Fig. AC).
  • Quantitative "real time" RT- PCR analysis disclosed a strong decrease in mRNA amounts of the neuroectodermal stem cell marker in HC-toxin treated BE(2)-C NB cells compared to controls.
  • the HDACI HC-toxin induces neuronal differentiation and loss of neuroectodermal stem cell marker expression at nanomolar concentration in NB cell lines.
  • Anchorage-independent growth potential and in vitro invasiveness were evaluated following treatment of BE(2)-C and WAC-2 cells with 20 nM HC-toxin or MeOH solvent control for 72 h. Analysis revealed that HC-toxin treated BE(2)-C and WAC-2 cells are characterized by a dose-dependent significant loss of anchorage- independent growth potential (Fig. 5 ⁇ 4) and of in vitro invasiveness in Boyden chambers (Fig. 5B). With respect to Fig. 5 A, after 12 and 24 days, respectively, plates were photographed and colonies stained with cell stain solution were counted. The experiment was repeated independently three times in duplicates with consistent results.
  • BE(2)-C NB cells treated with 15 or 20 nM HC-toxin displayed a significantly stronger hypophosphorylated RB (pRB) fraction compared to MeOH treated cells (Fig. 6A). This effect persisted at all time points investigated. Similar effects were induced by HC-toxin in Kelly, LA-N-I, and NGPNB cells (data not shown). A strong dose-dependent induction of p21 cipl/waf l at 24 h of HC-toxin treatment was observed in all NB cell lines investigated. After 12O h of HC toxin treatment, p21 cipl/waf"1 was down-regulated by HC-toxin treatment and therefore appeared to be contra-regulated to p27 tapl .
  • pRB hypophosphorylated RB
  • PHSFs human skin fibroblasts
  • Western analysis revealed an induction of acetylation of histone 4 after 6 h comparable to that seen in neuroblasts (Fig. IA, upper panel).
  • Fig. IA the number of viable PHSFs during HC-toxin treatment remained unchanged (Fig. IA).
  • Fig. IA harvested cells were evaluated for number and viability by trypan blue exclusion. Results presented are means ⁇ SDs of three independent experiments performed in triplicates. Induction of histone 4 acetylation (panAc-H4) by HC-toxin is shown in the upper panel.
  • Fig. 1C Shown at right are exemplarily FACS profiles of solvent control and HC-toxin treated cells after 72 h of exposure. All parameters investigated remain unaltered after 72 h of treatment. Striped areas represent S phase cells. Accordingly, phosphorylation of RB remained unaltered despite HC-toxin treatment (Fig. 1C). With respect to Fig. 1C, unsynchronized actively growing PHSFs were treated with 20 nM HC-toxin or MeOH solvent control, and protein lysates were harvested after 24 h of treatment for Western analysis with ⁇ -actin serving as loading control. In conclusion, HC- toxin promotes no detectable effects upon PHSFs.
  • HDAC inhibitors Tumor-selective action of HDAC inhibitors involves TRAIL induction in acute myeloid leukemia cells. Nat Med, 11: 77-84, 2005.

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Abstract

La présente invention porte sur le traitement d'un sujet atteint d'une tumeur de petites cellules bleues arrondies par un inhibiteur de l'histone désacétylase, en particulier par un inhibiteur qui est la toxine de Helminthosporium Carbonum. La toxine de Helminthosporium carbonum inverse le dosage nanomolaire dans les cellules de tumeur de l'état malin à bénin. La conversion à l'état bénin est associée à l'activation de réseaux de suppression de tumeur de rétinoblastome. La stimulation des voies de suppression de tumeur présente une nouvelle approche à une thérapie de tumeur de petites cellules bleues arrondies.
PCT/EP2007/061206 2006-10-20 2007-10-19 Inhibiteur de l'histone désacétylase, la toxine d'helminthosporium carbonum, pour la suppression de qualité maligne de cellules de neuroblastome WO2008046909A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020102599A1 (fr) * 2018-11-14 2020-05-22 Vanderbilt University Traitement de rétinoblastome intraoculaire avec des inhibiteurs de la modification d'histone
CN109912686A (zh) * 2019-03-13 2019-06-21 北京大学深圳研究生院 一种靶向hdac的稳定多肽类抑制剂及其用途
CN109912686B (zh) * 2019-03-13 2022-07-05 北京大学深圳研究生院 一种靶向hdac的稳定多肽类抑制剂及其用途

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